The major goal of our research has been to elucidate the molecular mechanisms whereby hormones regulate the growth and differentiation of mammalian cells using the mouse mammary gland as a model. The synergistic actions of lactogenic hormones, insulin, prolactin and glucocorticoid induce the functional differentiation of mammary epithelium by stimulating the expression of milk protein genes and other types of gene involved in secretory activities. Our previous studies have indicated that the hormonal stimulation of milk protein gene expression involves induction of positive transcription factors, which enhance the gene transcription via binding to specific promoter regions. We have identified and characterized some of these transcription factors. We also have extended our studies by examining the mechanisms involved in hormonally-induced expression of non-milk protein genes such as UDP-Glc NAc dolichol phosphate N-acetylgucosamine-1-phosphate transference (GPT). GPT is the enzyme, which catalyzes the transfer of Glc NAc-1-P from UDP-GlcNAc to Dol-P to form Glc NAc-P-P-Dol and thus plays a key role in the biosynthesis of aspargine-linked glycoproteins during lactogenesis. The activity of GPT in the mammary gland increases substantially during the period of lactation. Transcription of the GPT gene in the gland, like that of casein gene, is stimulated by insulin, glucocorticoid, and prolactin. Transient transfection of various GPT promoter/reporter constructs identified a distal negative regulatory region and deletion of this region resulted in enhanced hormonal induction with no effect on basal promoter activity. Gel-shift assays performed with mammary gland nuclear extracts indicated that the binding activity to the negative regulatory region was predominant in virgin stage as compared with lactating stage. Organ culture studies revealed that the binding activity in virgin mouse tissue was markedly decreased in the presence of insulin, glucocorticoid and prolactin. DNase I footprinting analysis identified two pentamer direct repeat motifs, AGGAA and GAAAC, within the negative regulatory region. Transcription from the promoter containing the mutated direct repeat was increased greatly, suggesting that these motifs serve to repress GPT gene expression. Thus, in contrast to casein gene, lactogenic hormones stimulate transcription of GPT gene by reducing the level of the repressor binding to the negative regulatory element in the promoter.